A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay

Area of Science:

• Biotechnology
• Analytical Chemistry
• Biochemistry

Background:

• Enzyme-linked immunosorbent assays (ELISA) are standard for biomolecule detection but often rely on antibodies.
• Antibody production can be complex and costly, presenting limitations for certain diagnostic applications.
• Developing alternative detection methods with high sensitivity and specificity is crucial for advancing diagnostics.

Purpose of the Study:

• To develop a rapid, sensitive, and antibody-free colorimetric assay for thrombin detection.
• To utilize aptamers for both capture and detection of thrombin in a sandwich assay format.
• To evaluate the performance of the aptamer-based assay against conventional ELISA.

Main Methods:

• A colorimetric sandwich-type assay was designed using aptamers.
• Aptamers immobilized on glass were used to capture thrombin.
• A second aptamer, conjugated with horseradish peroxidase (HRP), facilitated colorimetric detection via 3,5,3',5'-tetramethylbenzidine (TMB).

Main Results:

• The assay demonstrated high linearity and sensitivity, detecting thrombin as low as 25 femtomolar (fM).
• The method provided rapid and reproducible thrombin detection.
• The aptamer-based assay showed recovery and accuracy superior to, or at least equal to, antibody-based ELISA.

Conclusions:

• A highly sensitive and rapid enzyme-linked aptamer assay for thrombin detection has been successfully developed.
• This aptamer-based assay eliminates the need for antibodies, simplifying the detection process.
• The assay exhibits performance comparable or superior to traditional ELISA, offering a promising alternative for thrombin quantification.